The present invention relates to methods for providing cytoplasmic male sterile Petroselinum crispum plants, cytoplasmic male sterile Petroselinum crispum plants and to seeds, cells, tissues and plant parts thereof.
The Sequence Listing submitted in text format (.txt) filed on May 21, 2015, named “2NJ60_5-20-2015.txt”, (created on May 20, 2015, 2 KB), is incorporated herein by reference.
Petroselinum crispum or parsley belongs to the Umbelliferae or Apiaceae family. Members of this family are flat leaf parsley (P. crispum var. neapolitanum), curly leaf parsley (P. crispum var. crispum) and Hamburg parsley or rooted parsley (P. crispum var. tuberosum).
Leaf type parsley is often used as a garnish and has a typical flavour caused by volatile oils like myristicin and flavonoids as apiin.
Rooted parsley has a long, fleshy, white taproot generally served as a vegetable or used in soups or stews. The root is rich in carotene, vitamins B2 and C. The leaves of the plant are prepared in a similar way as of leaf type parsley. Rooted parsley is especially popular in Germany, Austria, Hungary and Russia. In Western Europe the crop is rediscovered, particularly in organic horticulture.
Other members of the Umbelliferae family are, for example, parsnip (Pastinaca sativa), carrot (Daucus carota L.), celery (Apium graveolens L.), fennel (Foeniculum vulgare Mill.), caraway (Carum carvi), anise (Pimpinella anisum), dill (Anethum graveolens) and coriander (Coriandrum sativum).
For cultivation of parsley, presently, only open pollinated crossbred races are available. As a result of the absence of male sterility or, for example, self-incompatibility of the crop, it is impossible to develop F1 hybrids.
F1 hybrids in general have advantages over open pollinated crops. According to the handbook “Principles of Plant Genetics and Breeding” by G. Acquaah (Blackwell Publishing, ISBN-13: 978-1-4051-3646-4; 2007; chapter 18) a hybrid cultivar is the F1 offspring of a planned cross between inbred lines. A critical requirement of hybrids production is that the parents are unidentical. This divergence gives hybrids their superior performance, due to the exploitation of heterosis or hybrid vigor. Hybrid vigor may be defined as the increase in size, vigor, fertility and overall performance of a hybrid plant over the average performance of the two parents (Ibid. pages 334-335).
The availability of F1 hybrids in parsley is considered advantageous over the present open pollinated crossbred races in a number of ways. F1 hybrids, as compared to the available open pollinated races, generally have an improved emergence, higher yields, more vigour and/or a high uniformity.
In general, F1 hybrids of a crop can be produced by the use of male sterile parent lines which are, inherently, unable to self-pollinate and are used as mother lines. An alternative, known for example from Brassica crops, is the use of self-incompatible plants which cannot self-pollinate and therefore make the production of F1 hybrids possible.
Male sterile parent lines provide control over the genetic composition of the progeny since the inheritance of traits is, in principle, fixed. Half of a progeny's nuclear genetic material, i.e. nuclear genome, originates from the male parent line (male fertile), while the other half of a progeny's nuclear genetic material, i.e. nuclear genome, originates from the female (male sterile) parent line from which the F1 seed is harvested.
Parsley plants, however, generally are both male and female (i.e. monoecious) and the absence of male sterile parent plants, such as in open pollinated crossbred races, makes a 100% reliable prediction of the genetic makeup, or (nuclear) genotype of the progeny impossible. At least some progeny, in the absence of male sterile plants, as a result of self-pollination, comprise the genetic material, or nuclear genome, originating from only one parent line.
The term hybrid, F1 hybrid or cybrid is often used in the art to denote that the plants concerned are heterozygous for at least some (commercially interesting) genotypic traits. Generally, a hybrid, F1 hybrid or cybrid is denoted in the art as comprising a heterozygous nuclear genome.
Male sterility in plants generally relates to cytoplasmic male sterility, wherein the determining genetic factor is located in the cytoplasm. For many plants it is demonstrated that this determining genetic factor is comprised in the mitochondria and generally encompasses a mutation in mitochondrial DNA.
Mitochondria are only inherited by the progeny through the egg cell or, in other words, by female inheritance, and not by the pollen, or, in other words, by male inheritance. Cytoplasmic male sterility is also designated in the art with the abbreviation CMS.
In some plants, besides cytoplasmic male sterility, also nuclear (male) sterility is observed. For nuclear (male) sterility, in contrast to cytoplasmic encoded sterility, a genetic factor in the nuclear genome, or the DNA of the nucleus, is responsible for the observed sterility. This nuclear sterility may relate to male, female or general sterility.
Both types of sterility, i.e. cytoplasmic male sterility and nuclear (male) sterility can easily be distinguished. Cytoplasmic male sterility is only inherited through the female parent line. In contrast, as an inherent result of its presence in the nucleus, nuclear sterility generally shows a Mendelian inheritance.
Through the use of CMS, cross pollination will result in 100% “pure” or “true” F1 hybrids. In contrast, using normal monoecious parsley plants, a certain percentage of self-pollination will occur in the next generation.
Fennel is a species within the Apiaceae family where CMS is a common trait, the use of F1 hybrids in fennel is generally known. In fennel, no genes are known which restore fertility; therefore it is an ideal source of CMS.
It was demonstrated that it was possible to obtain a cross between parsley and fennel. In other words, a cross wherein one parent line, the pollinator, is parsley and the other parent line is CMS fennel. However, this cross appeared to be very inefficient; instead of the expected amount of at least 2500 seeds only a very limited amount of seeds could be obtained by flowering these plants in the presence of blowflies. Most of these seeds were non-viable as well. After germination of the seed the desired progeny was indeed obtained.
Although, eventually aided by embryo rescue, this progeny obtained can be crossed further with parsley, thereby diluting the amount of fennel nuclear genome, after several generations of backcrossing an inbred generation, made by self-pollination, is required.
Using an inbred generation, it is in principle possible to select progeny having the desired phenotype (a parsley genome without fennel characteristics). Despite this, a large number of generations will still comprise small amounts of fennel DNA, causing undesired characteristic or traits in the hybrid obtained. It cannot be predicted at which point in time this remaining fennel DNA will be eliminated from the nuclear genome.
However, with the present case of cytoplasmic male sterile (CMS) fennel, the required inbred generation of a fennel-parsley hybrid is not possible because the plants needed for this surely are male sterile, therefore making an inbred generation technically impossible.
Considering, amongst others, the above advantages of F1 hybrids, such as an improved emergence, higher yields, more vigour and/or a high uniformity, there is a need in the art to provide cytoplasmic male sterile (CMS) Petroselinum crispum plants. Accordingly, it is an object, amongst other objects, of the present invention to fulfil this need.